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Changes of cellular expression of mRNA for tropoelastin in the intraembryonic arterial vessels of developing chick revealed by in situ hybridization.
No full textThe pattern of expression of tropoelastin mRNA in the arterial tree of developing chick has been studied by in situ hybridization. Significant hybridization was noted in 5.5-day embryos in the region of the truncus arteriosus where aorta and pulmonary artery had newly separated. The activation of expression then propagated centrifugally and longitudinal gradients of mRNA decreasing from the heart to the periphery were established. For almost two-thirds of the embryonic period, the hybridization signal was rather uniform over the entire wall of the arterial vessels. Later, however, its distribution varied depending on the type of artery (elastic or muscular) and on the developmental stage. A radial gradient of tropoelastin mRNA expression decreasing in the in-out direction was formed in elastic arteries. This was first seen in the pulmonary artery (15-day chick embryos) and became detectable in the vessels of the general circulation only much later (2 weeks after hatching). The appearance of the radial gradient was followed by a general reduction of mRNA synthesis. In muscular arteries radial gradients were also established, but had, however, an opposite polarity; in small arteries a ring of hybridization was evident at the media-adventitia border. The results indicate that the expression of the tropoelastin gene in cells of the arterial wall is finely regulated, depending on the coordinates in the arterial tree, the type of artery and the organ supplied
Detection of elastin by immunoelectronmicroscopy. A comparison of different procedures.
No full textElastin components have been identified in chick aorta by different immunoelectronmicroscopic procedures (peroxidase-antiperoxidase, immunoferritin and immunogold) using affinity purified antibodies to chick tropoelastin. The PAP method used in a preembedding procedure stained the outer portion of amorphous elastin and the microfibrils very intensively. The surface of the cells was also slightly stained. On the contrary immunogold labelling on Epon or Lowicryl embedded sections produced a strong decoration only of amorphous elastin, while microfibrils remained almost completely unlabelled. The result is not due to loss of antigenicity of microfibrils during embedding, since similar data were obtained with immunoferritin in a preembedding procedure. Experiments performed under different stringency conditions showed that the products of the peroxidase reaction diffuse and redistribute in the tissue, indicating that the positive staining of microfibrils and cell surface is an artifact. The value of different immunological reagents and procedures in studying the fine mapping of elastin components is discussed
Multiple binding reactivities of an IgG1 mouse monoclonal antibody raised against the extracellular matrix glycoprotein gp 115.
No full textAn IgG1 mouse monoclonal antibody that recognized five different polypeptides from chick and man is described. The antibody was raised against the elastin associated chick glycoprotein gp 115. In addition to the immunizing antigen, antibody 106D6 bound to chick smooth muscle cell myosin and to the alpha, beta and gamma chains of human fibrinogen. Cross reaction was demonstrated by radioimmunobinding, immunofluorescence and immunoblotting experiments. The immunoblotting assays were carried out using identical amounts of the various antigens to exclude the possibility that relevant quantitative differences in their concentration might result in non-specific binding
Monoclonal antibodies against chick gp 115, a matrix glycoprotein with broad distribution.
No full textHybridoma cell lines were generated producing monoclonal antibodies to chick gp 115, a 115,000-dalton glycoprotein widely distributed in the connective tissue. The specificity of the antibodies was determined by indirect radioimmunobinding: the extent of binding was a function of i) antigen and ii) antibody concentration; iii) inhibition of binding of radiolabelled antibody by unlabelled antibody and iv) among many known extracellular collagenous or noncollagenous glycoproteins tested only gp 115 gave a strong positive binding reaction. The antibodies were used for indirect immunofluorescence and a strong staining reaction was detected in all blood vessels, around smooth muscle cells in several organs, and in the connective matrix of other tissues such as the liver, and the lung. Based on the competition of binding of [125I]-labeled purified antibody by unlabeled antibodies, two separate epitopes were identified on gp 115. Further analysis of the localization of the epitope was obtained by CNBr cleavage and partial digestion of gp 115 with Staphylococcus aureus V8 protease and alpha-chymotrypsin digestion. Following CNBr cleavage a major fragment of Mr = 35,000 was recognized by 4 monoclonal antibodies, and fragments of comparable Mr were detected following V8 protease and alpha-chymotrypsin digestion
Identification of a TGF-beta responsive element in the human elastin promoter.
No full textIn a previous report (Marigo, V., Volpin, D., and Bressan, G. M. (1993) Biochim. Biophys. Acta 1172, 31-36) it was shown that the elastin promoter contains a region mediating transcriptional activation by TGF-beta in aorta cells, but not in tendon fibroblasts from chick embryos. In this paper we have identified the sequence responsible for this effect by a combination of CAT assays with mutant constructs, DNase I footprinting and electrophoretic mobility shift assays. This TGF-beta responsive element binds different nuclear proteins in chick embryo aorta and tendon cells. Whereas association of the aorta protein(s) to the element is necessary for TGF-beta activation, binding of the tendon protein(s) has apparently no effect on promoter stimulation by the cytokine
Ultrastructural immuno-localization of tropoelastin in the chick eye.
No full textImmuno-gold labeling at the electron-microscopy level was used to investigate the distribution of tropoelastin in the chick eye. Intense staining was found in the amorphous part of mature elastic fibers in different regions of the organ. In elaunin fibers, both the amorphous core and the surrounding microfibrils were clearly labeled. In addition, reactive sites were detected in the oxitalan fibers of the stroma- of the cornea and in Descemet's membrane, which showed a gradient of reactive sites increasing from the center toward the periphery. Oxitalan fibers of the stroma often fused with Descemet's membrane; the pattern of immunological staining suggested a continuity between the two structures. In the ciliary zonule, labeling for tropoelastin was observed in discrete areas on the bundles of microfibrils. The results show a complex structural organization of elastic tissue; this may be important in endowing the various parts of the eye with different mechanical properties
Transcriptional activation of the a1(VI) collagen gene during myoblast differentiation is mediated by multiple GA boxes.
No full textDuring differentiation of ClC12 myoblasts in vitro, expression of alpha 1(VI) collagen mRNA was transiently stimulated severalfold. Promoter assays on cells transfected with chloramphenicol acetyltransferase (CAT) chimeric constructs have identified a region of the alpha 1(VI) a collagen promoter that increases CAT activity about 8-fold during differentiation. The region, which overlaps with transcription initiation sites, was shown to contain three protected segments (A, B, and C) in DNase I footprinting assays. The contact points between nuclear factors and the protected segments were determined by methylation interference assay and included the sequence GGGAGGG (GA box) in all segments. Experiments in which CAT constructs were cotransfected with double-stranded oligonucleotides containing the GA box suggested that this motif was necessary for induction. Transfections with deletion constructs of the natural promoter and with minipromoters made of three copies of A, B, or C showed that the elements have inducing activity and that elements C and, to a lower extent, B are stimulatory for basal transcription, whereas the contribution of A in this process is limited. Electrophoretic mobility shift assays with nuclear extracts from C2C12 cells indicated that the three GA box-containing elements bound several transcription factors, including Sp1. Comparison of the properties of the bands shifted under different experimental conditions (presence of 10 mM EDTA, heating of the nuclear extracts, addition of different concentrations of competitor oligonucleotides) established that A, B, and C probes form nine, eight and five main retarded complexes, respectively, and indicated that nuclear factors binding to C and B are subsets of proteins binding to A. UV cross-linking assays identified several peptides (seven with probe A, six with B, And five with C) in the range of 150-32 kDa. Comparison of the gel retardation pattern obtained with nuclear extracts from proliferating and differentiating cells revealed a particular increased intensity of two retarded bands. The data establish that multiple GA boxes mediate induction of the alpha 1(VI) collagen promoter during myoblast differentiation and suggest the attractive hypothesis that the effect may be related to variations of expression of transcription factors binding to these motifs
Mapping of binding sites for monoclonal antibodies to chick tropoelastin by recombinant DNA techniques.
No full textA fusion molecule consisting of the entire coding sequence of mature chicken tropoelastin preceded by 14 amino acids of the signal peptide and 9 amino acids of vector origin has been expressed in a recombinant bacterial system and purified. The molecule has been used as immunogen for the production of hybridomas. Monoclonal antibodies which bound specifically the immunogen were also reactive with tropoelastin purified from chick aorta and stained elastic fibers in aorta sections by immunofluorescence. The region of tropoelastin containing the antigenic determinant recognized by each antibody has been identified by a recombinant DNA expression strategy based on the use of cDNA clones spanning different portions of the coding sequence. It could be shown that several antibodies were directed against unique epitopes; among these, a group of antibodies bound specifically to the sequence (PGVGV)n. Other antibodies were found to recognize antigenic determinants present more than once in the molecule. The monoclonal antibodies thus characterized will be useful reagents in studying the function of the different domains of tropoelastin
SREBP contributes to induction of collagen VI transcription by serum starvation
No full textCollagen VI is a main extracellular matrix protein whose mutation is linked to myopathic diseases. In myoblasts and other cell types, collagen VI gene transcription peaks during cell-cycle exit that precedes differentiation, upon serum withdrawal or confluence. To get insight into this transcriptional regulation, we characterized a growth arrest responsive region (GARR) in the Col6a1 promoter responsible for this effect. In this work, we identify sterol regulatory element binding protein (SREBP) as a GARR binding protein and provide evidence that SREBP contributes to induction of Col6a1 transcription in serum free conditions. Furthermore, our data unveil a previously unexpected link between extracellular matrix production and LDL signalin
Fine mapping of tropoelastin-derived components in the aorta of developing chick embryo.
No full textAffinity-purified antitropoelastin antibodies have been used to localize tropoelastin-derived components in aortas from chick embryos of different age by immunoelectron microscopy. Staining in the matrix is first noted at day 3 associated with irregular bundles of filaments resembling microfibrils, in the absence of amorphous elastin deposits. Amorphous material, which rapidly accumulates at later stages, is heavily labelled, while surrounding microfibrils are only poorly labelled. By contrast, a more intense staining of microfibrils persists in regions in which amorphous material is not morphologically evident. These observations indicate that the initial accumulation of elastin requires microfibrils, while the two components are not in close association in the subsequent growth of the amorphous core of the fibre. Intracellular staining is evident in the secretory apparatus of the cell and in peripheral large vesicles. Differentiated cells also show regions of close contact with elastic fibres in which immunological staining for elastin is very close to the cell membrane
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